The present invention relates to a glucose sensor which facilitates rapid and simplified quantitative analysis of a specific component contained in a sample with high accuracy. More specifically, the present invention relates to a method for stabilizing glucose dehydrogenase whose coenzyme is pyrrolo-quinoline quinone and a glucose dehydrogenase composition obtained by the stabilizing method.
Conventionally, a variety of biosensor have been proposed as a system facilitating simple quantitation of a specific component contained in a sample solution without requiring dilution or agitation of a sample solution. The following is a known example of such biosensor (Japanese Laid-Open Patent Publication No. Hei 2-062952).
The blosensor disclosed in this prior art is produced by the steps of forming an electrode system including a working electrode, a counter electrode and a reference electrode on an electrically insulating base plate using a known screen printing method or the like and subsequently forming immediately on this electrode system an enzyme reaction layer containing a hydrophilic polymer, an oxidoreductase, and an electron acceptor.
Upon a dropwise addition of a sample solution containing a substrate over the enzyme reaction layer of the biosensor thus produced, the enzyme reaction layer dissolves in the sample solution and the substrate in the sample solution is oxidized by the enzyme. At that time, the electron acceptor is reduced. After enzyme reaction is completed, the reduced electron acceptor is electrochemically reoxidized. The concentration of the substrate in the sample solution can be determined based on the oxidation current produced by the reoxidation reaction.
In principle, the biosensor as described above permits measurements of various materials if a suitable enzyme corresponding to the substrate of an analyte is selected.
For example, if glucose oxidase is the selected enzyme, then a glucose sensor for measuring a glucose concentration in the sample solution can be obtained.
The biosensor of the above structure normally accommodates the enzyme in the dried state. However, the enzyme is susceptible to degeneration when exposed to water in air for a long time, because it is essentially composed of protein which is readily degraded. In the extreme, the enzyme is exposed to a risk of losing the enzyme activity.
Therefore, long-term preservation of sensors after their production may result in a loss of activity of the enzyme and a depletion of necessary enzyme for reacting with the substrate. This may lead to a noncommensurable sensor response current to the substrate concentration.
In general, introduction of a sample solution containing a 0% substrate can produce some degree of sensor response current (hereinafter referred to as xe2x80x9cblank valuexe2x80x9d). One cause of such blank value may be induction of electrode reaction due to an accumulation of ions contained in the sample solution dissolving the reaction layer on the surface of the electrode system formed on the base plate. A large blank value can serve as a factor for impairing the correlation between response current and substrate concentration, rendering it impossible to make precise quantitative analysis of the substrate.
Therefore, securing an environment where the enzyme can retain the enzyme activity for a long term in the vicinity of the enzyme is key to the provision of a biosensor demonstrating excellent stability against preservation and producing a low blank value. It is also important to secure an environment, which produces a minimal and negligible blank value, around the surface of the electrode system on the base plate. It is also necessary to realize smooth transfer of both electron and substrate during enzyme reaction so as to enhance sensor response.
One conventional countermeasure for solving the above-mentioned problems is an inclusion of an additive such as phosphoric acid in the reaction layer.
In order to produce a high performance glucose sensor, on the other hand, glucose dehydrogenase whose coenzyme is pyrrolo-quinoline quinone (hereinafter abbreviated to xe2x80x9cPQQ-GDHxe2x80x9d) has conventionally been used as the enzyme. If PQQ-GDH is included as the enzyme, the resultant glucose sensor inherently has a characteristic feature of complete freedom from any adverse influence of dissolved oxygen in blood or the like on the enzyme reaction, because oxygen plays no role in the catalytic action of PQQ-GDH. Therefore, measurement values obtained from such glucose sensor are also free of variations due to oxygen partial pressure in the sample solution. This means that a high performance sensor will result from the use of PQQ-GDH as the enzyme.
However, the use of PQQ-GDH as the enzyme has a drawback that even inclusion of an additive such as phosphoric acid as exemplified before in the reaction layer can not help the resultant biosensor to lower the blank value sufficiently and to demonstrate sufficiently high stability against preservation.
In view of the above-mentioned problems, a primary object of the present invention is to provide a high performance glucose sensor demonstrating high stability against preservation and producing a low blank value. Other objects of the present invention are to provide a method for stabilizing PQQ-GDH and a glucose dehydrogenase composition obtained by the stabilizing method.
The glucose sensor in accordance with the present invention comprises an electrically insulating base plate, an electrode system including at least a working electrode and a counter electrode formed on the base plate, and a reaction layer which is formed in contact with or in the vicinity of the electrode system and contains at least PQQ-GDH, wherein the reaction layer further contains at least one additive selected from the group consisting of phthalic acid, a phthalate, maleic acid, a maleate, succinic acid, a succinate, triethanol amine, a triethanol amine salt, citric acid, a citrate, dimethyl glutaric acid, 2-(N-morpholino)ethane sulfonic acid, a 2-(N-morpholino)ethane sulfonate, tris(hydroxymethyl)glycine, a tris(hydroxymethyl)glycine salt, tris(hydroxymethyl)aminomethane, a tris(hydroxymethyl)aminomethane salt, imidazole, and collidine.
In a preferred mode of the present invention, the enzyme is coated with the additive.
The present invention also relates to a method for stabilizing glucose dehydrogenase for use in glucose sensors, wherein at least one additive is added to PQQ-GDH, the additive being selected from the group consisting of phthalic acid, a phthalate maleic acid, a maleate, succinic acid, a succinate, triethanol amine, a triethanol amine salt, citric acid, a citrate, dimethyl glutaric acid, 2-(N-morpholino)ethane sulfonic acid, a 2-(N-morpholino)ethane sulfonate, tris(hydroxymethyl)glycine, a tris(hydroxymethly)glycine salt, tris(hydroxymethyl)aminomethane, a tris(hydroxymethyl)aminomethane salt, imidazole, and collidine.
The present invention further relates to a glucose dehydrogenase composition for use in glucose sensors, the composition containing PQQ-GDH and at least one additive selected from the group consisting of phthalic acid, a phthalate, maleic acid, a maleate, succinic acid, a succinate, triethanol amine, a triethanol amine salt, citric acid, a citrate, dimethyl glutaric acid, 2-(N-morpholino)ethane sulfonic acid, a 2-(N-morpholino)ethane sulfonate, tris(hydroxymethyl)glycine, a tris(hydroxymethyl)glycine salt, tris(hydroxymethyl)aminomethane, a tris(hydroxymethyl)aminomethane salt, imidazole, and collidine.
In an aspect of the invention, the glucose sensor comprises an electrically insulating base plate, an electrode system including at least a working electrode and a counter electrode formed on said base plate, and a reaction layer which is formed in contact with or in the vicinity of said electrode system. The reaction layer contains: at least one stabilizer selected from the group consisting of a metal salt, an organic acid, a protein, and a sugar and a derivative thereof; a glucose dehydrogenase whose coenzyme is pyrrolo-quinoline quinone; and a buffer.
In another aspect of the invention, the method for stabilizing glucose dehydrogenase for use in glucose sensors includes adding a stabilizer and a buffer to glucose dehydrogenase whose coenzyme is pyrrolo-quinoline quinone.
In yet another aspect of the invention, a glucose dehydrogenase composition for use in glucose sensors contains: at least one stabilizer selected from the group consisting of a metal salt, an organic acid, a protein, and a sugar and a derivative thereof; a glucose dehydrogenase whose coenzyme is pyrrolo-quinoline quinone; and a buffer.
The buffer is selected from the group consisting of maleic acid, a maleate, succinic acid, a succinate, triethanol amine, a triethanol amine salt, citric acid, a citrate, dimethyl glutaric acid, 2-(N-morpholino)ethane sulfonic acid, a 2-(N-morpholino)ethane sulfonate, tris(hydroxymethyl)glycine, a tris(hydroxymethyl)glycine salt, tris(hydroxymethyl)aminomethane, a tris(hydroxymethyl)aminomethane salt, imidazole or colicin.
The stabilizer can be a metal salt selected from the group consisting of a calcium salt, CaCl2, a strontium salt and a manganese salt; an organic acid selected from the group consisting of xcex1-ketoglutaric acid, malic acid, fumaric acid, gluconic acid, cholic acid and deoxycholic acid; a protein selected from the group consisting of bovine serum albumin, egg albumin and gelatin; a sugar or a derivative thereof selected from the group consisting of a monosaccharide and a derivative thereof, a disaccharide, and a derivative thereof, an oligosaccharide and a derivative thereof, and a polysaccharide and a derivative thereof; a monosaccharide selected from the group consisting of glucose, fructose, galactose, mannose, xylose, inositol, monnitol, sorbitol, ribitol, glucosamine and deoxyglucose, or a derivative thereof; a disaccharide selected from the group consisting of sucrose, lactose, maltose and trehalose, or a derivative thereof; an oligosaccharide selected from the group consisting of malt triose, maltosyl cyclodextrin, xcex1-cyclodextrin, xcex2-cyclodextrin and xcex3-cyclodextrin, or a derivative thereof; a polysaccharide selected from the group consisting of dextrin, amylose, glycogen, inulin and Ficoll, or a derivative thereof.
While the novel features of the invention are set forth particularly in the appended claims, the invention, both as to organization and content, will be better understood and appreciated, along with other objects and features thereof, from the following detailed description taken in conjunction with the drawings.